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Medical biotechnology
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
To achieve maximum purity in a single step, affinity purification can be performed by using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine that allows selective attachment to a carrier protein, such as keyhole limpet hemocyanin (KLH), during development and to support purification. The antibody-containing media are then incubated with the immobilized antigen, either in batches or as the antibody is passed through a column, where it is selectively retained while impurities are removed. An elution with a low pH, high salt elution buffer is then used to recover the purified antibody from the support. To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis. The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis (CE) can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.
Preparation monoclonal β-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique
Published in Preparative Biochemistry & Biotechnology, 2020
Luhuai Shi, Tao Yu, Miner Luo, Hong Wang
Based on the monoclonal antibody (mAb) of ZEN (named 1G4) produced by our lab, we generated monoclonal β-type anti-idiotype antibodies of zearalenone derived from 1G4-KLH (Keyhole Limpet Hemocyanin) conjugates. These antibodies could imitated ZEN to establish a strong binding property with anti-ZEN mAb that proved to be a good ZEN substitute. By using anti-ZEN mAb and ZEN monoclonal Ab2β, an innocuous ELISA quantitative detecting technique was developed. Without use standard ZEN, this detecting technique is environment-friendly, cheap, fast and notoxic. The results of the study showed that the innocuous ELISA quantitative detecting technique had similar specificity and repeatability to classical assay.
Progress on electrochemical sensors for the determination of heavy metal ions from contaminated water
Published in Journal of the Chinese Advanced Materials Society, 2018
Xiangzi Dai, Shuping Wu, Songjun Li
Immunosensors are compact analytical devices based on specific antigen–antibody interactions and in which the immunochemical reactions are either directly or indirectly detected by means of a transducer. In electrochemical immunosensors, the event of immunochemical reactions is converted into an electrical signal such as an electric current, a voltage difference or a resistivity change.[21,74–76] The main principle of electrochemical immunosensor for the quantification of heavy metal ions is the change in the current on electrode surface due to oxidation and reduction of adsorbed metal ions. An ultrasensitive electrochemiluminescent (ECL) competitive immunoassay for mercury (II) was developed based on CdSe QDs, gold nanoparticles (GNPs) and specific monoclonal antibody (mAb) against Hg(II).[77] GNPs as substrate and electron transfer accelerator could load more number of coating antigen and magnify the electrochemical signal. Based on this method, Jing et al. developed a cheap and selective GNPs/Ovalbumin-MNA-CH3 Hg/mAb-QDs immunosensor based on the specific mAb against Hg(II). Competitive immunoassay was applied for the detection of Hg(II), and the ECL assay process is depicted in Figure 5(a). The actual size of the thioglycolic acid (TGA) modified CdSe QDs was about 4.6 nm (Figure 5(b)). The possible ECL mechanism could be expressed by the cyclic voltammetry (CV) of the immunosensor (Figure 5(c)). Furthermore, at the range of 0.01 to 50 ng/mL, the logarithm of the mercury (II) concentration change is linear to the decrease of the ECL intensity, with a much lower than previous methods detection limit of 2.6 pg/mL. The immunosensor also exhibit great selectivity to the mercury (II), the result was manifested in the (Figure 5(c)). This ECL immunoassay which applied GNPs as substrate combined with specific mAb detecting Hg(II), is presented for the first time. In addition, Zhu et al. had specifically generated and selected measured environmental uranium with an antibody-based sensor based on recombinant single-chain variable fragment antibodies (scvF).[78] The obtained scvF was complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP) using genetic material obtained from the spleen cells of rabbits immunized with UO22+—DCP conjugated to keyhole limpet hemocyanin. Recombinant antibody library and phage-displayed antibodies were obtained by amplifying and cloning immunoglobulin light chain and heavy chain genes into the phagemid pSD3. The limit of detection to UO22+ was 2.2 nM, which is below the United States Environmental Protection Agency (US EPA) action limit of 126 nM. In the real sample, the recovery ranged from 84.9% to 124.5% and average sample recovery was 98.89%.